Electrochemiluminescence method of detecting an analyte in a liquid sample and analysis system

ABSTRACT

An electrochemiluminescence method of detecting an analyte in a liquid sample and a corresponding analysis system. An analyte in a liquid sample is detected by first providing a receptacle containing a fluid comprising protein coated magnetic microparticles to a stirring unit. Stirring of the fluid is necessary since the density of the microparticles is usually higher than the density of the buffer fluid. Thus the microparticles tend to deposit on the bottom of the receptacle leading to an aggregation of the microparticles because of weak interactions. To obtain representative measurements a homogeneous distribution of the microparticles in the buffer fluid is necessary to ensure a constant concentration of microparticles for each analysis cycle. It is further necessary to provide disaggregation of the microparticles, which is also realized by stirring the fluid. Stirring is conducted with a rotational frequency that is adapted to the amount of fluid to be stirred.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/561,284, filed Sep. 5, 2019, which is a divisional continuation ofU.S. patent application Ser. No. 14/968,118, filed Dec. 14, 2015, whichis a continuation of International Patent Application No.PCT/EP2014/060149, filed 16 May 2014, which claims the benefit ofEuropean Patent Application No. 13172826.3, filed 19 Jun. 2013, thedisclosures of which are hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

The present disclosure relates to the field of medical technology anddiagnostics and, in particular, to a method of detecting an analyte in aliquid sample by effecting luminescence, such as by using a luminescenceimmunoassay, and a respective analysis system.

BACKGROUND

Numerous methods and systems have been developed for the detection andquantitation of analytes of interest in biochemical and biologicalsubstances. Methods and systems that are capable of measuring traceamounts of microorganisms, pharmaceuticals, hormones, viruses,antibodies, nucleic acids and other proteins are of great value toresearchers and clinicians.

A significant body of art has been developed based upon the well-knownbinding reactions, e.g., antigen-antibody reactions, nucleic acidhybridization techniques, and protein-ligand systems. The high degree ofspecificity in many biochemical and biological binding systems has ledto many assay methods and systems of value in research and diagnostics.Typically, the existence of an analyte of interest is indicated by thepresence or absence of an observable “label” attached to one or more ofthe binding materials.

Chemiluminescent assay techniques where a sample containing an analyteof interest is mixed with a reactant labeled with a chemiluminescentlabel have been developed. The reactive mixture is incubated and someportion of the labeled reactant binds to the analyte. After incubation,the concentration of the label in either or both fractions can bedetermined by chemiluminescent techniques. The level ofchemiluminescence determined in one or both fractions indicates theamount of analyte of interest in the biological sample.

Electrochemiluminescent (ECL) assay techniques are an improvement onchemiluminescent techniques. They provide a sensitive and precisemeasurement of the presence and concentration of an analyte of interest.In such techniques, the incubated sample is exposed to apotentiostatically or galvanostatically controlled working electrode inorder to trigger luminescence. In the proper chemical environment, suchelectrochemiluminescence is triggered by a voltage or current impressedon the working electrode at a particular time and in a particularmanner. The light produced by the label is measured and indicates thepresence or quantity of the analyte.

In accordance with electrochemiluminescence binding reaction analysis(ECL-BBA) a detectable complex is produced, whose concentrationconstitutes a measure of the analytic result sought. A markingsubstances (label) capable of effecting an ECL-reaction is coupled to abinding reagent specific for the analyte, e.g., an antibody. The speciescomprising the marking substance and the binding reagent is designatedas a label conjugate.

When such a substance is subjected to a suitable electrical potential ona voltammetric working electrode, it emits light which can be measuredphotometrically. A second electrochemically active substance, designatedas a co-reactand, normally contributes to this reaction. In practice,primarily a ruthenium complex (ruthenium-tris [bipyridyl]) is used asECL-label in combination with TPA (tripropylamine) as co-reactand. Thetwo electrochemically active substances are both oxidized upon voltageapplication to the electrode. Subsequent loss of a proton will turn theTPA into a strongly reducing species. The subsequent redox reactionbrings the ECL-label into an excited state from which it returns to theground state with the emission of a photon. The ECL-label reaction ispreferably a circular reaction so that a single label molecule emits aplurality of photons after application of a voltage to the electrode.

The ECL-marked complex molecules characteristic for the analysis arefixed to magnetic microparticles (beads). In practice, magnetizedpolystyrene beads having a diameter of typically 2 to 3 micrometers areused. Fixing is effected by means of a pair of specific biochemicalbinding partners. The pair streptavidin biotin has turned out to beparticularly advantageous. The beads are streptavidin-coated, to which abiotinylated antibody will bind.

The beads with the bound marked complex are introduced into themeasuring cell of a measuring apparatus. The cell is equipped withelectrodes which are necessary for generating the electrical fieldrequired for triggering the ECL-reaction. The beads are drawn onto thesurface of the working electrode in the magnetic field of a magnetdisposed below the working electrode. Since this typically occurs inflow-through cells with continuously flowing sample fluids, the magneticdeposition of the beads is designated as “capturing”. An electricpotential required for triggering the ECL-reaction is then applied tothe working electrode and the resulting luminescence light is measuredusing a suitable optical detector. The intensity of the luminescencelight is a measure for the concentration of the number of labeledantibodies coupled to the beads on the surface of the working electrodewhich, in turn, is a measure of the concentration of the analyte in thesample. A calibration allows calculation of the sought concentrationfrom the measured luminescence signal.

A plurality of different variations of this type of ECL-BB A-method havebeen discussed and described in the literature.

SUMMARY

It is against the above background that the embodiments of the presentdisclosure provide certain unobvious advantages and advancements overthe prior art. In particular, the inventors have recognized a need forimprovements in luminescence methods for detecting an analyte in aliquid sample and an analysis system.

In accordance with one embodiment of the present disclosure, anelectrochemiluminescence method of detecting an analyte in a liquidsample is provided, the method comprising: providing a receptaclecontaining a fluid comprising protein coated magnetic microparticles toa stirring unit; acquiring a signal being indicative of an amount of thefluid contained in the receptacle; determining a rotational frequencyfor the stirring unit dependent on the amount of fluid in thereceptacle, the rotational frequency being proportional to the amount offluid; stirring the fluid for a predefined period of time by applyingthe previously determined rotational frequency; taking a portion of thefluid containing the protein coated magnetic mi-croparticles from thereceptacle, thereby reducing the amount of the fluid contained in thereceptacle; mixing a portion of the liquid sample with the portion ofthe fluid comprising the protein coated magnetic microparticles and witha marker; incubating the mixture comprising the analyte, the proteincoated magnetic microparticles, and the marker in an incubator;transporting a portion of the mixture from the incubator to ameasurement cell; applying a magnetic field to the measurement cell formagnetic adhesion of the protein coated magnetic microparticles to aworking electrode of the measurement cell; applying an excitation energyfor causing luminescence; measuring of the luminescence for acquisitionof a measurement signal, and generating an output signal beingindicative of the presence of the analyte in the liquid sample using themeasurement signal.

In accordance with another embodiment of the disclosure, anelectrochemiluminescence analysis system for detecting an analyte in aliquid sample is provided comprising: a stirring unit for stirring afluid containing magnetic microparticles provided in a receptacle; ameasuring unit being operable to generate a signal indicative of anamount of fluid in the receptacle; an extraction component forextracting a portion of the fluid containing the protein coated magneticmicroparticles from the receptacle; an incubator for receiving a liquidcomprising the analyte, the portion of magnetic microparticles and amarker for marking the analyte, the marker being capable of effectingluminescence upon application of excitation energy; a trigger componentfor applying the excitation energy for causing the luminescence; and anacquisition component for measuring the luminescence the acquisitionunit being operable to provide a measurement signal. The analysis systemfurther comprises a data processing unit configured to determine arotational frequency for the stirring unit using the signal indicativeof the amount of fluid in the receptacle, the rotational frequency beingproportional to the amount of fluid; control the stirring unit to stirthe fluid for a predefined period of time by applying the previouslydetermined rotational frequency; and generate an output signal beingindicative of the presence of the analyte in the liquid sample using themeasurement signal.

These and other features and advantages of the embodiments of thepresent disclosure will be more fully understood from the followingdetailed description taken together with the accompanying claims. It isnoted that the scope of the claims is defined by the recitations thereinand not by the specific discussion of features and advantages set forthin the present description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the presentdisclosure can be best understood when read in conjunction with thefollowing drawings, where like structure is indicated with likereference numerals and in which:

FIG. 1 is a block diagram of an analysis system in accordance with oneembodiment of the disclosure;

FIG. 2 is a diagram illustrating the ECL-BBA technique in accordancewith one embodiment of the disclosure;

FIG. 3 is a block diagram of an analysis system comprising a roboticcomponent in accordance with another embodiment of the disclosure;

FIG. 4 is a block diagram of a rotor for receiving receptacles inaccordance with an embodiment of the disclosure; and

FIG. 5 is a schematic of a stirring unit in accordance with anembodiment of the disclosure.

Skilled artisans appreciate that elements in the figures are illustratedfor simplicity and clarity and have not necessarily been drawn to scale.For example, the dimensions of some of the elements in the figures maybe exaggerated relative to other elements to help improve understandingof the embodiments of the present disclosure.

DETAILED DESCRIPTION

In some embodiments of the present disclosure an analyte in a liquidsample is detected by first providing a receptacle containing a fluidcomprising protein coated magnetic microparticles to a stirring unit.Stirring of the fluid comprising protein coated magnetic microparticlesis necessary since the density of the microparticles is usually higherthan the density of the buffer fluid. Thus the microparticles tend todeposit on the bottom of the receptacle leading to an aggregation of themicroparticles because of weak interactions. To obtain representativemeasurements a homogeneous distribution of the microparticles in thebuffer fluid is necessary to ensure a constant concentration ofmicroparticles for each analysis cycle. Besides homogenization it isfurther necessary to provide disaggregation of the microparticles aswell, which is also realized by stirring the fluid comprising themicroparticles.

In this context the term “to stir” is to be understood as “to mixsomething by making circular movements in it using an object suitablefor mixing”.

Stirring the fluid comprising protein coated microparticles has severaladvantages over other ways and means to disaggregate the microparticles.For example, the microparticles may be disaggregated using supersonicsound. Supersonic sound may however cause damage of the protein coatingof the microparticles. Another possibility may be vortex mixing of thefluid containing the microparticles, which may however cause severe foamgeneration that is detrimental for representative measurements asdiscussed below. A third possibility may be the use of extensional flowcaused by a piston pipettor. The use of a piston pipettor for mixing thefluid may however cause mechanical stress for the protein coatedmicroparticles which may lead to damage of the coating.

To provide disaggregation of the microparticles a certain amount ofenergy dependent on the amount of fluid comprising the microparticles tobe stirred has to be applied to the fluid. The amount of energy appliedto the fluid during the stirring process depends on the rotationalfrequency of the stirrer and the amount of time during which the fluidis stirred.

In a next method step a signal being indicative of the amount of fluidcontained in the receptacle is acquired. The signal may for example beacquired by reading an RFID tag of the receptacle or other memory or bymeasuring the filling level of the receptacle as described below. Onemay also weigh the receptacle containing the fluid and knowing theweight of the receptacle itself and the density of the fluid comprisingthe magnetic microparticles could determine the volume of the fluidcontained in the receptacle.

Dependent on the previously determined amount of fluid contained in thereceptacle a rotational frequency for the stirring unit is determined,for example by looking up a table comprising information on whichrotational frequency to use for which amount of fluid. The appliedrotational frequency should be high enough to provide for short timeperiods for stirring the fluid. However the rotational frequency shouldnot be too high, because a too high rotational frequency might causefoam generation which in turn may lead to unwanted effects. For example,extraction of predefined and repeatable amounts of fluid may not bepossible if the fluid is at least partially foamed.

After stirring the fluid for a predetermined period of time by applyingthe previously determined rotational frequency, a portion of the fluidcontaining the protein coated magnetic microparticles is taken from thereceptacle, thereby reducing the amount of fluid contained in thereceptacle. The portion of the fluid comprising the magneticmicroparticles is then mixed with a portion of the liquid samplecomprising the analyte and a marker, and the mixture is incubated in anincubator. A portion of the mixture is then transported to a measurementcell, where a magnetic field is applied for magnetic adhesion of theprotein coated magnetic microparticles to a working electrode of themeasurement cell.

After application of an excitation energy for causing luminescence theluminescence is measured for acquisition of a measurement signal. Usingthe measurement signal an output signal being indicative of the presenceof the analyte in the liquid sample is generated.

An “analyte” as understood herein is a component of the liquid sample tobe analyzed, e.g., molecules of various sizes, proteins, metabolites andthe like.

A “liquid sample” as understood herein encompasses a biological samplesuch as any kind of tissue or body fluid having been derived from ahuman or any other organism. In particular, a biological sample can be ablood-, serum-, plasma-, urine-, cerebral-spinal fluid-, orsaliva-sample, or any derivatives thereof.

The term “luminescence” as understood herein encompasses any kind ofluminescence such as radiation-induced luminescence, chemiluminescenceand electrochemiluminescence, in particular ECL-BBA.

The term “luminescence immunoassay” as understood herein encompasses anyimmunoassay that produces an optical signal, i.e., a luminescence signalthat indicates the presence of a particular analyte in a liquid sample.

The method described above is configured so that individual analysisruns can be carried out with constant expenditure of time. During aseries of analysis cycles the amount of fluid comprising the magneticmicroparticles contained in a receptacle may change. Thus the amount ofenergy to be applied to the fluid during stirring has to be adapted tothe amount of fluid contained in the receptacle. However, using themethod steps described above the rotational frequency of the stirrer canbe adapted to the amount of fluid to be stirred, such that the time forstirring can be held constant for each stirring process.

This facilitates the automation of the previously described analysisprocedure. In most common analysis systems employing a stirring unit therotational frequency of the stirrer is the same for each analysis cycle,irrespective of the amount of fluid to be stirred. Thus the rotationalfrequency of the stirrer has to be adapted such that even for a minimalamount of fluid contained in the receptacle no foam is generated by thestirring process. However if the receptacle contains a large amount offluid stirring with the previously described low rotational frequencywill consume considerably more time than stirring with a rotationalfrequency that has been adapted to the amount of fluid to be stirred andthus will slow down the analysis process.

In some embodiments acquiring a signal being indicative of the amount offluid contained in the receptacle is conducted using a pipetting probethat is also used for extracting a portion of fluid from the receptacle.This may have the advantage that a signal being indicative of the amountof fluid contained in the receptacle may be generated while a portion offluid is extracted from the receptacle. Thus, two method steps can becarried out simultaneously thereby reducing the expenditure of time ofan analysis cycle.

In some embodiments the pipetting probe may comprise a capacitive sensorfor acquiring a signal being indicative of the amount of fluid containedin the receptacle. For example, the capacitive sensor may be designedsuch that it responds to changes of the humidity of its environment. Thecapacitive sensor may be located at the tip of the pipetting probe suchthat a measurement signal of the capacitive sensor is produced once thetip of the pipetting probe comes into contact with the surface of thefluid contained in the receptacle. To obtain the filling level of thereceptacle from the previously described measurement signal, one may forexample lower the pipetting probe from a known starting height inpredefined steps. After each step the voltage or any other indicator ofthe capacitive sensor is measured. Once the tip of the pipetting probecomes into contact with the fluid a value of the measured indicator willchange. Since the starting height of the pipetting probe is known andthe step size of the lowering process is known as well, one can easilydetermine the filling level of the receptacle.

In some embodiments determining the rotational frequency for thestirring unit may comprise providing a signal being indicative of theamount of fluid contained in the receptacle to a data processing unit.The data processing unit may for example comprise a table wherein thetable comprises information on which rotational frequency to use for acertain amount of fluid contained in the receptacle. Even though it maybe possible to calculate the values for such a table using the laws ofphysics and for example employing a numerical method, according to atypical embodiment the data may also be determined empirically. Once thedata processing unit determined the rotational frequency to use bylooking up the table, the data processing unit provides a second signalbeing indicative of the rotational frequency appropriate for the amountof fluid that has previously been determined.

In another aspect the present disclosure relates to an analysis systemfor detecting an analyte in a liquid sample.

In some embodiments the analysis system may comprise a stirring unit forstirring a fluid containing magnetic microparticles provided in areceptacle and a measuring unit being operable to generate a signalindicative of an amount of fluid in the receptacle. For extracting aportion of the fluid containing the protein coated magneticmicroparticles from the receptacle, the analysis system may furthercomprise an extraction component. To initiate a transfer of the liquidcomprising the analyte, the portion of magnetic microparticles and amarker for marking the analyte, the analysis system may further comprisean incubator. The marker for marking the analyte typically is capable ofeffecting luminescence upon application of excitation energy. To obtaina measurement signal the analysis system may further comprise a triggercomponent for applying the excitation energy for causing theluminescence and acquisition component for measuring the luminescence,the acquisition unit being configured/operable to provide a measurementsignal and a data processing unit being adapted to determine anappropriate rotational frequency for the stirring unit using the signalindicative of the amount of fluid in the receptacle and to generate anoutput signal being indicative of the presence of the analyte in theliquid sample using the measurement signal.

In this case the term “adapted to” with reference to the data processingunit of the analysis system includes that the data processing unit isconfigured to carry out the described method steps.

It is understood that one or more of the aforementioned embodiments ofthe disclosure may be combined as long as the combined embodiments arenot mutually exclusive.

In order that the embodiments of the present disclosure may be morereadily understood, reference is made to the following examples, whichare intended to illustrate the disclosure, but not limit the scopethereof.

FIG. 1 shows an analysis system 100 for detecting an analyte in a liquidsample. The analysis system 100 comprises an incubator 102 for receivinga liquid 104 that is a mixture of an aliquot of the liquid sample and amarker for marking the analyte, such as of a luminescence immunoassay.

The analysis system 100 comprises a reservoir 106 that contains theco-reactant of the electrochemical reaction causing the luminescence.The incubator 102 and the reservoir 106 are coupled to a measurementcell 108 of the analysis system by a pipe system 110 through which aportion of the liquid 104 and the co-reactant can flow into themeasurement cell 108.

The measurement cell 108 comprises a cell body 112 that has a conduit114 for receiving a portion of the liquid 104 and of the co-reactantthrough the pipe system 110. The measurement cell 108 has a magneticcomponent 116, such as a permanent magnet, for providing a magneticfield in the measurement cell. The magnetic component 116 may be coupledto an actuator 118 for rotating the magnetic component 116 to and fromthe conduit 114 in order to switch on or off the magnetic field withinthe conduit.

The magnetic component 116 is positioned below a working electrode 120that is coupled to a voltage source 122. An excitation area 124 isformed in the conduit 114 within the magnetic field caused by themagnetic component 116 on the working electrode 120.

Luminescence that is caused in the excitation area 124 by theapplication of excitation energy, i.e., the application of a voltaictrigger pulse on the working electrode 120, is measured by means of anoptical sensor, such as a photomultiplier 126. The optical sensor issensitive within a certain frequency range such that it provides ameasurement signal, such as a luminescence signal caused byautoluminescent molecules that may be present in the measurement cell,provided that the luminescence is within the sensor's frequency range.

The photomultiplier 126 is positioned opposite to the excitation area124 over a window formed by counter electrodes 128 of the workingelectrode 120 through which the luminescence photons and any interferingphotons caused by the excitation energy impinge on the photomultiplier126. A resultant time resolved measurement signal 130 is provided fromthe photomultiplier 126 to a control unit 132 of the analysis system100.

After a measurement has been performed the liquid contained within theconduit 114 is removed into a liquid waste container 134 and themeasurement cell 108 is regenerated for a subsequent acquisition of ameasurement signal.

The control unit 132 is coupled to the voltage source 122 in order tocontrol the voltage source 122 to apply the trigger signal to theworking electrode 120. The control unit 132 is also coupled to theactuator 118 for controlling the actuator 118 to switch on and off themagnetic field by moving the permanent magnet correspondingly.

Further, the control unit 132 may be coupled to a “sipper unit”, i.e., apump 136, for extracting a portion of the liquid 104 from the incubator102 and a portion of the co-reactant from the reservoir 106 as well asfor removing the liquid from the measurement cell 108 and regenerationof the measurement cell. In addition, the control unit 132 may becoupled to additional robotic components such as a pipetting station(cf., embodiment of FIG. 3 ).

The measurement cell 108 may be adapted for performing ECL-BBA usingvarious luminescence immunoassays.

For example, the liquid 104 may contain a mixture of an aliquot of theliquid sample, streptavidin coated magnetic particles, biotinylatedantibodies and ruthenylated antibodies to form a so-called “sandwich”whereas the co-reactant contained in the reservoir 106 istripropylamine. Hence, magnetic particles 138 with a bound label flowinto the conduit 114. The magnetic particles 138 are immobilized on theworking electrode 120 when the magnetic field is switched on. Next, thetrigger pulse is applied on the working electrode 120 to cause theelectrochemiluminescence in accordance with the ECL-BBA technique.

The control unit 132 has an electronic memory 140 for storing data 142that describes which rotational frequency for a stirring unit to use fora certain amount of fluid contained in a receptacle 168 (cf., FIG. 3 ).In the embodiment considered here the data 142 is stored in a lookuptable or database table and will be discussed with reference to FIG. 3 .

The control unit 132 has at least one electronic processor 144 forexecution of program modules 146 and 148. Program module 146 is executedby the processor 144 for acquisition of the measurement signal 132whereas the program module 148 is executed by the processor 144 forevaluation of the acquired measurement signal 132.

The control unit 132 has an interface 150 for coupling a display 152 oranother human-machine-interface to the control unit 132. The interface150 may be implemented as a graphical user interface for displaying awindow 154 for a user's selection of one of the luminescenceimmunoassays supported by the analysis system 100 as well as a window156 for displaying a result of the analysis.

The result of the analysis performed by the analysis system 100 may beoutput as tabular data as depicted in FIG. 1 wherein the column Aindicates the analyte to be detected and the column C indicates theconcentration of the analyte that has been detected.

In operation, a user selects one of the luminescence immunoassayssupported by the analysis system 100 by entering a respective selectioninto the window 154. The analysis of the liquid sample is started byexecution of the program module 146 such that the pump 136 is controlledto transport a portion of the liquid 104 and of the co-reactant into theconduit 114.

Next, the actuator 118 is controlled to flip the permanent magnet into aposition such that its magnetic field is applied to the conduit 114 forimmobilization of the magnetic particles with their bound labels on theworking electrode 120. Next, the voltage source 122 is controlled toapply the trigger pulse onto the working electrode for excitation of theluminescence such that the measurement signal 130 results.

The measurement signal 130 is acquired by sampling the output of thephotomultiplier 126 over a given period of time, such as 2 seconds afterapplication of the trigger pulse by the voltage source 122, fortime-resolved measuring of the luminescence.

The data samples that constitute the measurement signal 130 are storedwithin the memory 140 of the control unit 132 and the program module 148is started for evaluation of the acquired measurement signal 130. Byexecution of the program module 148 the concentration C of the analyte,if any, in the liquid is determined by means of the measurement signal130.

Next, the pump 136 is controlled by the control unit 132 for removingthe liquid from the conduit 114 and regeneration of the measurement cell108.

FIG. 2 is illustrative of the “sandwich” that is formed within theincubator 102 and to which a trigger pulse is applied within theexcitation area 124 on the working electrode 120. In the embodimentconsidered here each of the magnetic particles 138 has a diameter ofabout 2.8 micrometers. The magnetic particle 138 is bound to abiotinylated antibody 158 of the immunoassay that is chosen depending onthe analyte 160 to be detected. A ruthenium complex (ruthenium-tris[bipyridyl]) bound to an antibody 162 that is chosen depending on theanalyte 160 is utilized as a luminescent label in the embodimentconsidered here.

Upon application of the voltaic trigger pulse an electrochemicalreaction is induced with the tripropylamine in accordance with theECL-BBA technique such that luminescence is caused.

FIG. 3 shows a further embodiment of an analysis system 100. Theanalysis system 100 has a rotor 164 for receiving receptacles, such assample tubes, where each sample tube contains a liquid sample. The rotor164 may hold a number of sample tubes for providing random access to apipettor 176.

The analysis system 100 has a second rotor 166 for receiving receptacles168 containing streptavidin-coated magnetic microparticles, receptacles170 containing biotinylated antibodies and receptacles 172 containingruthenylated antibodies. The second rotor may be implemented as areagent disk as shown in FIG. 3 for providing access of the pipettor 176to the various reagents contained in the receptacles 168, 170 and 172.The second rotor 166 further comprises a stirring unit 178 that may forexample be mounted in the center of the reagent disk such that thestirring unit 178 can provide disaggregation of the streptavidin-coatedmagnetic microparticles contained in the receptacle 168 by stirring thefluid contained in receptacle 168 (cf., FIGS. 4, 5 ). Control unit 132controls the stirring unit 178 thereby causing the stirring unit 178 tostir the fluid contained in receptacle 168 with a rotational frequencythat is adapted to the amount of fluid contained in the receptacle 168.

The analysis system 100 has a robotic component for providing a mixtureto the incubator 102. In the embodiment considered here the roboticcomponent is controlled by the control unit 132 and comprises apipetting station 174 having the pipettor 176.

In operation, the control unit 132 determines rotational frequency forthe stirring unit 178 to stir the fluid in receptacle 168. Therotational frequency may for example be determined by the control unit132 by determining the amount of fluid contained in receptacle 168 andlooking up a table comprising information on which rotational frequencyto use for which amount of fluid. The amount of fluid contained in thereceptacle 168 may for example be determined from the filling level ofthe receptacle 168 as described below.

Once an appropriate rotational frequency for the stirring unit 178 hasbeen determined the stirring unit is controlled by the control unit 132to stir the fluid in receptacle 168 with the determined rotationalfrequency for a predetermined amount of time.

The control unit 132 then controls the pipettor 176 to extract analiquot of the liquid sample from one of the sample tubes that are heldby the rotor 164 and to extract portions of the streptavidin-coatedmagnetic particles, the biotinylated antibodies and the ruthenylatedantibodies from the receptacles 168, 170 and 172, respectively, in orderto provide the mixture that is then put into the incubator 102 forincubation during a predetermined amount of time, such as 9 to 27 min.

Upon extracting a portion of the streptavidin-coated magneticmicroparticles the amount of fluid contained in receptacle 168 may bedetermined. For example the pipettor may comprise capacitive meanssuitable for providing a measurement signal once the pipette tip touchesthe surface of the fluid contained in receptacle 168. If the startingheight of the pipette tip is known and the change of height caused by avertical actuator is logged during lowering, the liquid level ofreceptacle 168 can be determined from the measurement signal of thecapacitive means and the corresponding height of the pipette tip.

The control unit 132 controls the “sipper”, e.g., the pump 136 (cf.,FIG. 1 ), such that the liquid mixture flows from the incubator 102 intothe conduit 114 of the measurement cell 108 together with theco-reactant, i.e., tripropylamine. Next, the control unit 132 controlsthe actuator 118, (cf., FIG. 1 ) to switch on the magnetic field andthen the voltage source 122 to apply the voltaic trigger pulse.

The resultant measurement signal 130 is acquired by the control unit 132by sampling the output of the photomultiplier 126.

FIG. 4 shows a rotor 166 for use in an analysis system as described withreference to FIG. 3 . The rotor 166 is designed for receiving e-packs180 comprising the receptacles 168 containing streptavidin-coatedmagnetic microparticles, the receptacles 170 containing biotinylatedantibodies and the receptacles 172 containing ruthenylated antibodies.The e-packs 180 are arranged to form a ring that can be rotatedclockwise or counterclockwise. The receptacle 168 containing thestreptavi-din-coated magnetic microparticles is located at the innermostposition of an e-pack 180.

Rotor 166 further comprises an e-pack shifter 182 for moving e-packs 180from an outer position 184 to an inner position 186. The e-pack shifter182 may for example employ a grappler 188 for moving the e-packs 180.For proper identification of a selected e-pack 180 the e-packs 180 areequipped with RFID tags that can be read or written by the RFIDreader/writer 190.

E-pack shifter 182 further comprises a stirring unit 178. The stirringunit 178 is located such that it may reach the innermost receptacle 168containing streptavidincoated microparticles of the e-pack 180 in innerposition 186 for stirring. In a central portion of the e-pack shifter182 a cleaning station 192 for the stirring unit 178 is located.

In operation, an e-pack 180 may be selected for processing by rotatingthe ring of e-packs 180 until the e-pack 180 to be processed has beenidentified by the RFID reader/writer 190. The e-pack 180 is then movedfrom the outer position 184 to the inner position 186 by the grappler188 of the e-pack shifter 182. The stirring unit 178 may then stir thefluid contained in the innermost receptacle 168 with a predeterminedrotational frequency to provide sufficient disaggregation of themicroparticles contained in the fluid.

The applied rotational frequency for the stirring unit 178 may forexample be provided to the stirring unit 178 by the control unit 132(not shown). For that purpose the control unit 132 may be coupled withthe RFID reader/writer 190 for exchange of information on which e-pack180 is currently in the inner position 186 for processing. The controlunit 132 may then look up a table comprising information on the amountof fluid contained in the corresponding receptacle 168 and then may lookup another table comprising information on which rotational frequency touse for the determined amount of fluid.

It is also possible to write information on the amount of fluid inreceptacle 168 of an e-pack 180 to the corresponding RFID tag using theRFID reader/writer 190. The control unit 132 then would simply have toread the information on the amount of fluid from the RFID tag using theRFID reader/writer 190 and look up a table comprising information onwhich rotational frequency to use for the determined amount of fluid.This would further have the advantage that the e-packs 180 could betrans-ferred from one analysis system to another without losing theinformation on the amount of fluid in receptacle 168 of the e-packs 180.

Once the stirring process has ended, the pipettor 176 of the pipettingstation 174 may extract portions of the streptavidin-coated magneticparticles, the biotinylated antibodies and the ruthenylated antibodiesfrom the receptacles 168, 170 and 172, respectively, in order to providethe mixture that is then put into the incubator 102 for incubation.After the portions have been extracted the e-pack 180 may be moved fromthe inner position 186 to the outer position 184 by grappler 188 andanother e-pack 180 may be selected by rotating the ring of e-packs 180.

Before a subsequent stirring process is started the stirring unit may becleaned using the cleaning station 192. To allow the stirring unit 178to reach both the sample tubes within the e-pack 180 in innermostposition 186 and the cleaning station 192 the stirring unit may forexample be pivot-mounted or mounted on a slide that can move thestirring unit from a mixing position to a cleaning position and viceversa.

FIG. 5 shows an exemplary embodiment of a stirring unit 178. Thestirring unit 178 comprises a stirrer 194 mounted on a shaft 196 that isdriven by a motor 198. The assembly of motor 198, shaft 196 and stirrer194 is mounted on a rod 200 such that the vertical position of thestirrer 194 can be varied. Block 202 below the stirrer 194 indicates theposition of a receptacle containing a fluid to be stirred.

The assembly of rod 200, motor 198, shaft 196 and stirrer 194 is mountedon a pivotable socket that is driven by shaft 204. Thus it is possibleto switch the stirring assembly between at least two positions: a firstposition in which the stirrer 194 is located above a receptaclecontaining a liquid to be stirred, and a second position in which thestirrer 194 and the shaft 196 may be cleaned, for example, by dippingstirrer 194 and shaft 196 into a cleaning liquid. Instead of mountingthe stirring assembly on a pivotable socket the stirring assembly couldalso be mounted on a slide to switch between the stirring and thecleaning position.

In operation, a receptacle is moved below the stirrer 194, for exampleby the grappler 188 of the e-pack shifter 182 as described withreference to FIG. 4 . Subsequently the stirring assembly comprisingstirrer 194, shaft 196 and motor 198, is lowered until the stirrer 194is completely immersed in the fluid. Then the motor 198 is controlled bya control unit 132 (not shown) to apply a rotational frequency to thestirrer appropriate for disaggregating microparticles contained in thefluid. The fluid is then stirred for a predetermined amount of time withthe predetermined amount rotational frequency to provide for sufficientdisaggregation of the microparticles. Once the stirring process iscompleted the stirring assembly is raised, until the stirrer is locatedabove the fluid at a sufficient height.

In a next step the shaft 204 may be operated to rotate the stirringassembly into a cleaning position in which the stirrer is located abovea receptacle comprising a cleaning liquid. After lowering the stirringassembly until the stirring assembly is fully immersed in the cleaningfluid the motor 198 may again rotate the shaft 196 and the stirrer 194thereby cleaning shaft 196 and stirrer 194. Upon raising the stirringassembly out of the cleaning liquid the stirring assembly may be rotatedback to the stirring position such that the stirring unit 178 is readyfor the next stirring procedure.

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed subject matteror to imply that certain features are critical, essential, or evenimportant to the structure or function of the embodiments disclosedherein. Rather, these terms are merely intended to highlight alternativeor additional features that may or may not be utilized in a particularembodiment of the present disclosure.

It is also noted that the terms “substantially” and “about” may beutilized herein to represent the inherent degree of uncertainty that maybe attributed to any quantitative comparison, value, measurement, orother representation. These terms are also utilized herein to representthe degree by which a quantitative representation may vary from a statedreference without resulting in a change in the basic function of thesubject matter at issue.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the embodiments describedherein without departing from the spirit and scope of the claimedsubject matter. Thus it is intended that the specification cover themodifications and variations of the various embodiments described hereinprovided such modifications and variations come within the scope of theappended claims and their equivalents. Numbered embodiments arepresented below.

LIST OF REFERENCE NUMERALS

-   Analysis system 100-   Incubator 102-   Liquid 104-   Reservoir 106-   Measurement cell 108-   A pipe system 110-   Cell body 112-   Conduit 114-   Magnetic component 116-   Actuator 118-   Working electrode 120-   Voltage source 122-   Excitation area 124-   Photomultiplier 126-   Counter electrode 128-   Measurement signal 130-   Control unit 132-   Container 134-   Pump 136-   Particle 138-   Memory 140-   Reference data 142-   Processor 144-   Program module 146-   Program module 148-   Interface 150-   Display 152-   Window 154-   Window 156-   Biotinylated antibody 158-   Analyte 160-   Ruthenylated antibody 162-   Rotor 164-   Rotor 166-   Receptacles 168-   Receptacles 170-   Receptacles 172-   Pipetting station 174-   Pipettor 176-   Stirring unit 178-   E-pack 180-   E-pack shifter 182-   Outer position 184-   Inner position 186-   Grappler 188-   RFID reader/writer 190-   Cleaning station 192-   Stirrer 194-   Shaft 196-   Motor 198-   Rod 200-   Block 202-   Shaft 204

What is claimed is:
 1. An electrochemiluminescence method of detectingan analyte in a liquid sample using a series of analysis cycles, themethod comprising, for each analysis cycle in the series of analysiscycles: providing a receptacle containing a fluid comprising proteincoated magnetic microparticles to a stirring unit, acquiring a signalbeing indicative of an amount of the fluid contained in the receptacle,determining a rotational frequency for the stirring unit dependent onthe amount of fluid in the receptacle, the rotational frequency beingproportional to the amount of fluid, stirring the fluid for a predefinedperiod of time by applying the previously determined rotationalfrequency, the predefined period of time being held constant for eachstirring process, wherein the stirring is performed for homogenizationand disaggregation of the microparticles, extracting a portion of thefluid comprising the protein coated magnetic microparticles from thereceptacle, thereby reducing the amount of the fluid contained in thereceptacle, mixing a portion of the liquid sample with the portion ofthe fluid comprising the protein coated magnetic microparticles and witha marker, incubating the mixture comprising the analyte, the proteincoated magnetic microparticles, and the marker in an incubator,transporting a portion of the mixture from the incubator to ameasurement cell, applying a magnetic field to the measurement cell formagnetic adhesion of the protein coated magnetic microparticles to aworking electrode of the measurement cell, applying an excitation energyfor causing luminescence, measuring of the luminescence for acquisitionof a measurement signal, and generating an output signal beingindicative of the presence of the analyte in the liquid sample using themeasurement signal wherein the rotational frequency of the stirring unitis maximally high but below a frequency that causes foaming.
 2. Themethod of claim 1, wherein extracting a portion of fluid from thereceptacle employs a pipetting probe, and wherein acquiring a signalbeing indicative of the amount of fluid contained in the receptacle isconducted using the pipetting probe.
 3. The method of claim 1, whereinacquiring a signal being indicative of the amount of fluid contained inthe receptacle employs a capacitive method.
 4. The method of claim 1,wherein determining the rotational frequency for the stirring unitcomprises: providing a first signal being indicative of the amount offluid contained in the receptacle to a data processing unit, in responseto providing the first signal to the data processing unit receiving asecond signal being indicative of the rotational frequency appropriatefor the amount of fluid from the data processing unit.
 5. The method ofclaim 4, wherein the data processing unit comprises a table, the tablecomprising information indicative of which rotational frequency to usefor a defined amount of fluid, wherein the data processing unit inresponse to receiving the first signal looks up the table for therotational frequency appropriate for the amount of fluid the firstsignal is indicative of and provides a second signal being indicative ofthe rotational frequency appropriate for the amount of fluid.
 6. Themethod of claim 5, wherein the data comprised in the table is determinedempirically.
 7. The method of claim 1, wherein stirring is onlyconducted prior to extracting fluid from the receptacle.
 8. The methodof claim 1, wherein the excitation energy is applied by applyingelectrical energy, radiation energy and/or chemical energy.
 9. Themethod of claim 1, the marker being capable of effectingelectrochemiluminescence, wherein an electrochemically active substancecontributes to an electrochemiluminescence reaction with the markerresulting in the luminescence.
 10. The method of claim 8, furthercomprising: carrying out a reaction sequence comprising at least onespecific biochemical binding reaction to form a complex as a result ofthe presence of the analyte in the liquid sample, the complex comprisingthe marker and said complex further being bound to a magneticmicroparticle, carrying out a detection cycle in a measuring cell havinga working electrode for determination of the presence of said analyte,said detection cycle comprising a capturing step during which thecomplex is contacted with a working electrode in such a manner that saidmicroparticle is attracted by the magnetic field of a magnetic componentpositioned on the side of the working electrode facing away from thesample, thereby being deposited on the surface of said working electrodefacing the sample, and applying a potential to the working electrode totrigger the electrochemiluminescence reaction of the marker with theelectrochemically active substance causing the luminescence of themarker to thereby determine the presence of the analyte in the liquidsample.
 11. The method of claim 1, wherein the luminescence is measuredusing an optical sensor, and wherein the measurement signal is acquiredby sampling an output signal of the optical sensor.
 12. Anelectrochemiluminescence analysis system for detecting an analyte in aliquid sample using a series of analysis cycles, the system comprising:a stirring unit for stirring a fluid comprising protein coated magneticmicroparticles provided in a receptacle, a measuring unit being operableto generate a signal indicative of an amount of fluid in the receptacle,an extraction component for extracting a portion of the fluid comprisingthe protein coated magnetic microparticles from the receptacle, anincubator for receiving a liquid comprising the analyte, the portion ofmagnetic microparticles and a marker for marking the analyte, the markerbeing capable of effecting luminescence upon application of excitationenergy, a trigger component for applying the excitation energy forcausing the luminescence, an acquisition component for measuring theluminescence the acquisition component being operable to provide ameasurement signal, a data processing unit being adapted to: determine arotational frequency for the stirring unit using the signal indicativeof the amount of fluid in the receptacle, the rotational frequency beingproportional to the amount of fluid, control the stirring unit to stirthe fluid for a predefined period of time by applying the previouslydetermined rotational frequency, the predefined period of time beingheld constant for each stirring process, wherein the stirring isperformed for homogenization and disaggregation of the microparticles,wherein the rotational frequency of the stirring unit is maximally highbut below a frequency that causes foaming, generate an output signalbeing indicative of the presence of the analyte in the liquid sampleusing the measurement signal.
 13. The analysis system of claim 12further comprising a database, the database comprising a tablecomprising information indicative of which rotational frequency to usefor a defined amount of fluid, wherein the data processing unit isfurther operable to access the database and look up table for therotational frequency appropriate for the amount of fluid the firstsignal is indicative of.
 14. The method of claim 2, wherein acquiring asignal being indicative of the amount of fluid contained in thereceptacle employs a capacitive method.
 15. The method of claim 2,wherein determining the rotational frequency for the stirring unitcomprises: providing a first signal being indicative of the amount offluid contained in the receptacle to a data processing unit, in responseto providing the first signal to the data processing unit receiving asecond signal being indicative of the rotational frequency appropriatefor the amount of fluid from the data processing unit.
 16. The method ofclaim 2, wherein stirring is only conducted prior to extracting fluidfrom the receptacle.
 17. The method of claim 2, wherein the excitationenergy is applied by applying electrical energy, radiation energy and/orchemical energy.
 18. The method of claim 2, the marker being capable ofeffecting electrochemiluminescence, wherein an electrochemically activesubstance contributes to an electrochemiluminescence reaction with themarker resulting in the luminescence.
 19. The method of claim 2, whereinthe luminescence is measured using an optical sensor, and wherein themeasurement signal is acquired by sampling an output signal of theoptical sensor.